Extensive literature has reported altered expression and/or mutation of the primary gap junction proteins (connexins, Cx) that couple myocytes in ventricles (Cx43) and atria (Cx40) among major causes of cardiac arrhythmias. We have found that numerous genes controlling a vast number of functional pathways are regulated in Cx43 null tissues and these regulations are accurately predicted from coordination with Cx43 gene (Gja1) in wildtypes. These findings suggest an additional etiology of the disease that is related to connexins but not necessarily to the intercellular coupling they provide. Our working hypothesis is that the genes encoding the heart rhythm determinants (HRD) are interconnected in connexin-dependent and connexin-independent transcriptomic networks whose topologies may change during development and exhibit slight differences between the two genders. Such regulatory networks, where linkage partners are rearranged and strength modified in disease, may explain downstream and parallel "ripples" of phenotypic change. We plan to verify this hypothesis, and build and characterize the atrial and ventricular webs of genes encoding hear rhythm determinants. In addition, we shall identify and quantify the connexin-dependent regulatory networks within these webs. Of particular interest will be to identify the gene pairs with strikingly similar or opposite coordination profiles because up-regulation of a similar one or down-regulation of an opposed are expected to compensate for the deficient expression of the counterpart (as "transcriptomic see-saws"). For these, we shall profile the atrial and ventricular transcriptomes of wildtype, Cx40 null and Cx43 conditional knockdown male and female mice at E19, 1, 2 and 4 weeks of their early life. The expression data will be also used to determine the expression variability and intercoordination of all quantifiable unigenes and study the age and gender dependence of the HRD gene webs. We have developed the Principal Gene Analysis by which to identify the heart rhythm determinants (HRD), build and characterize the webs of their encoding genes. The "see-saw" model will be tested by comparing the transcriptomes of cultured cardiomyocytes in which expression of either Cx43 or of certain positive "see-saw" partners are knocked-down through siRNA treatment. Thus, this study is expected to reveal new organizational principles of the heart transcriptome and the role of gap junction genes in this organization, with the long-term goal to open novel therapeutic horizons in the treatment of arrhythmia. PUBLIC HEALTH RELEVANCE: Gap junctions between cardiac muscle cells provide the channels for intercellular current flow that assures propagation of contraction throughout the heart. Increasing evidence attributes numerous cardiac arrhythmias to altered gap junctions and the proteins of which they are composed. We hypothesize that this role of gap junction gene expression is due in part to linkage to expression of other genes that affect the cardiac rhythm, and we propose to use gene expression profiling from microarrays to achieve a comprehensive understanding of arrhythmia and to generate hypotheses testable by more focused methods. We plan to profile the gene expressions in atria and ventricles of male and female wildtype mice and mice lacking the main cardiac gap junction proteins at four time- points during evolution to the adult state to identify and quantify on a genome-wide scale the connexin- related transcriptomic determinants of arrhythmia, analyzing their gender dependence and maturation. A major contribution will consist in identifying candidate genes whose manipulation might restore the normal cardiac rhythm based on their similar or opposed expression coordination with cardiac connexins in the sampled transcriptome and to that of other genes whose alteration generates arrhythmia. The study will reveal new organizational principles of the heart transcriptome, with the long- term goal to open novel therapeutic horizons in the treatment of arrhythmia. Our project has the unique feature of describing and quantifying the Cx40- and Cx43-dependent Gene Regulatory Networks of the heart rhythm, with the long term goal to open novel therapeutic horizons in cardiology.